Bioconjugation and Detection of Lactosamine Moiety using α1,3-Galactosyltransferase Mutants That Transfer C2-Modified Galactose with a Chemical Handle

Glycan moieties of glycoproteins and glycolipids, by mediating protein–carbohydrate (1–4) and carbohydrate-carbohydrate interactions (5–7), play an important role in several cellular processes and are implicated in human diseases and cancer. The terminal sugar residues of the glycans are often important for their specific protein–carbohydrate interactions, for example only the sialyated glycans have high affinity for CD22, a regulatory molecule that prevents the overactivation of the immune system and the development to autoimmune diseases (4). Similarly, de-sialyated glycans with terminal galactose bind mostly to galectin molecules. Pathophysiological role of glycans, as seen in the presence of the unusual short glycans, such as Tn carbohydrate in Core 1 glycan or elongated glycans, such as the polylactosamine moiety in N-glycans, have been associated with cancer (8–10). Therefore, the ability to detect different terminal sugar moieties is vital for determining the association of these moieties with different cellular states. The detection of these sugar moieties have been achieved mostly through lectin- or antibody-binding studies. These methods, however, lack high specificity and sensitivity. In limited situations, modified sugars with a unique chemical handle have been used for in vivo incorporation in the metabolic pathway to enable the detection of sugar residues on the glycoconjugates of a cell (11–14). Consequently a highly specific and sensitive chemoenzymatic method to detect β-GlcNAc residue at the nonreducing end of glycans using the mutant enzyme of β1,4-galactosyltransferase, Y289L β4Gal-T1, has been recently developed (15–18). A mutation of the residueTyr289 to Leu289 in bovine β4Gal-T1 broadens the sugar donor specificity of the enzyme in such a way that the mutant enzyme, Y289L β4Gal-T1, transfers GalNAc from UDP-GalNAc to a sugar acceptor with the same efficiency as Gal from UDP-Gal (15). The mutant enzyme also transfers galactose analogues that have a chemical handle at the C2 position of the galactose and are similar in size and shape to the N-acetyl group, such as 2-keto-galactose (16) or GalNAz (Gal-2-NH-CO-CH2-N3) from their respective UDP-sugar substrates (17). The transfer of these galactose analogues with a unique chemical handle at the C2 position, such as a keto or azido group, by the mutant enzyme enables one to detect of GlcNAc residue using a sensitive chemiluminescence method, or to conjugate variety of molecules in a site-specific manner via GlcNAc residue (17, 18). Similarly, the polypeptide-α-GalNAc-T2, which transfers GalNAc from UDP-GalNAc to the side chain hydroxyl group of a Thr/Ser residue of a specific peptide sequence, has been shown to transfer the modified galactose with a chemical handle, either 2-keto-galactose or GalNAz, from its respective UDP-sugar (19). It has been shown that only a few residues in the sugar donor-binding pocket of glycosyltransferases determine the sugar donor specificity of these enzymes and that mutation of these residues can alter their sugar donor specificity (15, 20–22). Consequently novel glycosyltransferases with broader donor specificities can be designed to transfer a sugar residue with a chemically reactive functional group to specific terminal moieties of glycoconjugates. To detect the terminal N-acetyllactosamine (LacNAc (Galβ1-4GlcNAc)) moiety that is the most prevalent sugar moiety found on cells, we engineered the bovine α3Gal-T enzyme, which has high acceptor specificity towards LacNAc, to transfer GalNAc or C2-modified galactose which can be used to detect LacNAc. Because of its high acceptor specificity, α3Gal-T enzyme has been previously used to transfer Gal from UDP-Gal to asialo-glycoprotein with the free LacNAc moiety at its N-linked glycans (23). The α3Gal-T enzyme transfers only galactose from UDP-Gal to the terminal galactose sugar of the LacNAc or lactose moiety forming the product with Galα1-3-linkage. It belongs to a family of α3Gal/GalNAc glycosyltransferases to which human blood group transferases A and B also belong. The blood group transferases A (α1,3-GalNAc-T-A) and B (α1,3-Gal-T-B) transfer GalNAc and Gal, respectively, to galactose moiety of the fucosylated LacNAc acceptor, Fucα1-2Galβ1-4GlcNAc. The amino acid sequence of transferase A and transferase B are identical (24, 25) except for four amino acids. Two of the four residues at positions 266 and 268, Leu266 and Gly268 in blood group A transferase, and Met266 and Ala268 in blood group B transferase, determine their respective sugar donor specificities. In fact, in blood group A and B enzymes a mutation of the residue at position 266 is sufficient to alter the donor specificity of these enzymes (20, 21). Since α3Gal-T exhibits high sequence similarity and nearly identical crystal structure with the blood group A and B enzymes, a mutation of the corresponding sugar donor-binding residue in α3Gal-T, specifically, His280, is expected to alter the sugar donor specificity of the α3Gal-T enzyme (26). Indeed, recently the mutations of the residues in the sugar donor-binding site of bovine α3Gal-T have been shown to enhance the GalNAc transfer from UDP-GalNAc (27, 28). In the present study we have rationally mutated not only His280 residue, but also its neighboring residues to alter its sugar donor specificity from Gal to GalNAc. We show that the mutants also selectively transfer the modified sugar 2-keto-Gal from its UDP derivative to LacNAc. This handle is used in biotinylation and subsequent detection of terminal LacNAc moiety in glycoproteins by chemiluminescence.